The overall goal of this proposal is to provide the principal investigator (PI) with the experiences and skills necessary to become an independent researcher studying basic mechanisms of corneal diseases. The scientific focus of this proposal is to gain a better understanding of the cellular and extracellular matrix (ECM) protein abnormalities caused by mutations in collagen VIII which lead to corneal endothelial cell (CEC) loss in Fuchs endothelial dystrophy (FED). Collagen VIII (COL8) plays a central role in the formation of Descemet membrane (DM), the specialized basement membrane of CECs, and mutations in the gene encoding the alpha2 chain of collagen VIII (COLSA2) cause FED in some patients. FED progresses over decades in humans and accounts for up to 29% of corneal transplants. Early stages of the disease are asymptomatic, and mildly affected tissues are not available. Thus, virtually no information exists about the early cellular and ECM changes leading to FED. Understanding these early pathogenic events could be increased greatly through the development of a reliable animal model of FED as well as detailed biochemical analysis of pathogenic COL8A2 mutations on collagen VIII function. The underlying hypothesis of this proposal is that mutations in the COL8A2 gene produce cellular and biochemical abnormalities which cause CEC loss in FED. Identifying these abnormalities should provide important insights into the pathogenesis of FED, which may suggest rational therapies for this important corneal disease. To address this hypothesis, two specific aims are proposed: Aim 1: To develop and characterize a mouse model of FED by introducing the R155Q COL8A2 mutation using gene targeting techniques. Aim 2: To investigate the effects of COLSA2 mutations known to cause FED on homotrimer and heterotrimer formation with alpha1 collagen VIII, the other COL8 subchain interacting with COL8A2 in vivo. In the course of the proposed research and selected didactic activities, the PI will gain invaluable training experience and mentoring in developing animal models of ocular disease and biochemical analyis of ECM proteins. Expertise in these areas is deemed invaluable for the PI who aspires to develop an independent research program studying basic mechanisms of corneal diseases.